The present invention relates to the discovery, identification and characterization of nucleotides that encode Di12, a protein that is overexpressed in breast cancer tissue. The invention encompasses Di12 nucleotides, host cell expression systems, Di12 proteins, fusion proteins, polypeptides and peptides, antibodies to the gene product, transgenic animals that express an Di12 transgene, or recombinant knock-out animals that do not express the Di12, and other compounds that modulate Di12 gene expression or Di12 activity that can be used for diagnosis, disease monitoring, drug screening, and/or the treatment of cancer disorders, including but not limited to breast cancer.
Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). Clinical data and molecular biologic studies indicate that cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.
Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saundorg Co., Philadelphia, pp. 68-79.) The neoplastic lesion may evolve clonally and develop an increasing capacity for growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host""s immune surveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).
Breast cancer is the most common form of malignancy in women. One in nine women in North America will develop breast cancer and about 30% of them will ultimately die from the disease (Boring et al., 1993, Cancer Statistics, CA-A Journal for Physicians, 43:7-26). The American Cancer Society estimated that in 1992 180,000 American women were diagnosed with breast cancer and 46,000 succumbed to the disease (Niederhuber, J. E. ed. Current Therapy in Oncology B. C. Decker, Mosby, 1993). A disturbing fact is the observation that breast cancer has been increasing at a rate of 3 percent per year since 1980 (Niederhuber, J. E., ed. Current Therapy in Oncology, B. C. Decker, Mosby, 1993).
The natural history of breast cancer is characterized by a long duration and marked heterogeneity within and among patients. Breast cancer is among the more slow-growing tumors, and the preclinical period before diagnosis and the clinical phases after initial treatment and even after the appearance of metastasis are measured in years and decades. Nevertheless, some patients have aggressive forms of the disease and do poorly. Other patients have such indolent forms of the disease that it is difficult to demonstrate that therapy has any effect on survival. During the long clinical phase, there is ample opportunity for clonal mutation and evolution, and it seems probable that individual patients may have multiple tumor clones, each with its own growth rate, propensity to metastasize, and sensitivity to drugs.
In both Europe and North America, early detection campaigns based on mass screening programs have been introduced in an effort to reduce mortality rates. Widespread use of these procedures has resulted in an increased frequency of detection of breast cancer, which in turn has contributed to a greater number of women with early stage disease (Harris et al., 1993, Cancer: Principles and Practice of Oncology, eds. De Vita, V. T., Hellman, S., and Rosenberg, S. A. (J.B. Lippincott, Philadelphia), 4th Ed., pp. 1264-232). Given the high degree of morphological heterogeneity of most breast cancers, it is at present still difficult to assess appropriate therapy and risk of recurrence for the majority of women-who present with early stage disease. The currently available criteria affecting prognosis are tumor size and grade, lymph node status, DNA ploidy and mitotic index, lymphovascular invasion, as well as estrogen receptor status (Harris et al., 1993, Cancer: Principles and Practice of Oncology, eds. De Vita, V. T., Hellman, S., and Rosenberg, S. A., J.B. Lippincott, Philadelphia, 4th Ed., pp. 1264-1332). These multiple parameters remain poorly correlated with the molecular events associated with a multi-step progression of malignancy, e.g., it has been recently well defined for colorectal cancers (Vogelstein et al., 1993, Trends in Genetics, 9:138-141; Kinzler et al., 1996, Cell, 87:159-170).
The recent discoveries that individuals with BRCA1 and BRCA2 mutations have a predisposition to breast cancer may now facilitate the detection of an early onset type disease for hereditary breast cancer (Easton et al., 1993, Cancer Surv, 18:95-1131; Miki et al., 1994, Science, 266:66-71; Tavtigian et al., 1994, Nature Gen, 12:333-337). The incidence of these cases however, is just 5-10% of all known breast cancers (Easton et al., 1993, Cancer Surv, 18:95-1131; Miki et al., 1994, Science, 266:66-71; Tavtigian et al., 1994, Nature Gen, 12:333-337). Thus, early and late stage specific tumor markers are desperately needed for more than 90% of sporadic forms of breast malignancies.
Tumor suppressor genes and oncogenes play critical roles in the development of malignancies. Amplification and activation of several oncogenes in primary breast tissues such as c-erb-b2 (HER-2/neu), c-MYC and INT2, have been previously implicated and subsequently evaluated for their prognostic potential (Allred et al., 1990, Proc Am Soc Clin Oncol, 9:23; Berns et al., 1992, Cancer Res., 52:1107-1113). Molecular factors such as growth factor receptors and hormone induced genes have also been investigated (Sainsbury et al., 1987, Lancet, 1:1398-1402; Iwase et al., 1994, Breast Cancer Res Treat, 33:83-88). Unfortunately, the expression of these genes is often limited to a small percentage of breast carcinomas only, and their predictive prognostic value remains unclear. The need for markers which would discriminate biological differences between primary and metastatic breast tumors and provide early diagnosis remains pressing. Such factors would assist in identifying individuals at risk for aggressive disease, and aid therapeutic decisions.
The present invention relates to the identification of novel genes whose expression pattern is upregulated in breast cancer tissues and cell lines, and the use of such genes and gene products as targets for diagnosis, drug screening and therapies.
In particular, the compositions of the present invention include nucleic acid molecules that encode the novel Di12 protein, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants which encode novel Di12 gone products. The compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention and hosts which contain such nucleic acid molecules. The compositions of the present invention also encompass the Di12 gene products, fusion proteins, and antibodies directed against such Di12 gene products or conserved variants or fragments thereof.
The nucleic acid sequence of the human Di12 gene (SEQ ID NO:1) is provided. The Di12 gene produces a transcript of approximately 1.35 kb and encodes a protein of 339 amino acids with a molecular weight of approximately 35 kD. Transcripts were detected in several breast cancer cell lines, as well as various normal tissues, including lung, kidney, pancreas and heart. The amino acid sequence of the predicted full length Di12 gene product does not contain a recognizable signal sequence, indicating that the Di12 gene product is an intracellular protein.
The present invention further relates to methods for the diagnostic evaluation and prognosis of breast cancer. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for detection of abnormal expression of the Di12 gene.
Antibodies to Di12 gene product of the invention can be used in a diagnostic test to detect the presence of Di12 gene product in body fluids. In specific embodiments, measurement of serum or plasma Di12 gene product levels can be made to detect or stage breast cancer, especially infiltrative ductal carcinoma.
The present invention also relates to methods for the identification of subjects having a predisposition to breast cancer. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of Di12 gene mutations, allelic variations and regulatory defects in the Di12 gene.
Further, methods and compositions are presented for the treatment of breast cancer. Such methods and compositions are capable of modulating the level of Di12 gene expression and/or the level of Di12 gene product activity.
Still further, the present invention relates to methods for the use of the Di12 gene and/or Di12 gene products for the identification of compounds which modulate Di12 gene expression and/or the activity of Di12 gene products. Such compounds can be used as agents to prevent and/or treat breast cancer. Such compounds can also be used to palliate the symptoms of the disease, and control the metastatic potential of breast cancer.